For billions of years, life has been stuck with just four letters of DNA's genetic code: adenine, cytosine, guanine, thymine, or
ACGT
.

Now there are six, according to a new study by scientists from The Scripps Research Institute.

The two new lab-created letters function in lab experiments almost exactly like natural DNA, said
Floyd E. Romesberg
, senior author of the paper. The study was
published
online Monday in the Proceedings of the National Academy of Sciences.

This is the first time that "unnatural" letters have been shown to work like natural DNA, Romesberg said. In
research
published last month, the Romesberg team had shown that these unnatural letters, dubbed d5SICS and dNaM, are faithfully copied like natural letters. But the new research actually demonstrates function.

There's a lot more to expanding the DNA alphabet than simply sticking new chemicals into DNA, Romesberg said.

"It has to be an independent letter to the alphabet," Romesberg said. "To do that, you have to be able to work it into different words. You have to be able to use it in lots of ways. And that's what no one had ever shown."

With the new letters, scientists can add any desired function to DNA, and the related molecule RNA, along with increasing its information-carrying capability, Romesberg said.

Pairing up

A quick primer: DNA letters pair up in a complementary fashion on the double helix to form a "base pair," like the natural letters. Just as A teams up with T and G teams up with C, d5SICS pairs with dNaM, although the two unnatural letters are held in place by different molecular forces than the natural ones.

These new letters can be used immediately in the laboratory, said Romesberg and Denis Malyshev, a graduate student in Romesberg's lab and first author of the paper.

For example, the unnatural letters function with a common tool of biotechnology called polymerase chain reaction, or PCR, that creates millions of exact copies of DNA.

"With linkers added to the unnatural nucleotides the unnatural base pair should find uses in different in vitro applications, where it could be used to site-specifically modify DNA or RNA with any functionality of interest, even if requiring massive and sequence-independent amplification," the study stated.

Artificial life

Eventually, the scientists say they'd like to perform the much more difficult task of incorporating the unnatural DNA into living cells, creating life forms that never could have existed in nature.

This quest for artificial life is being pursued by several teams of scientists, including one led by famed gene pioneer J. Craig Venter.

Venter's team
synthesized the genome
of a bacterium in 2008, proving that the complete genetic code of a living creature could be made in the laboratory. In 2010, they put together a bacterial genome with natural and artificial DNA sequences, then
inserted
the synthetic DNA into a bacterium that had been deprived of its own DNA.

The bacterium's molecular machinery worked with the DNA, or "booted up," as the scientists put it. This creation marked the first time scientists had designed a new life form, however, most of its genes are natural. So it is not a wholly synthetic life form.

Romesberg said Venter's approach differs from his, and that of a friendly rival to his,
Steven A. Benner
. Venter is designing new genes and combinations of genes, but using the same four letters found in nature, Romesberg said.

Romesberg and Benner's teams are designing new letters for the DNA alphabet that do not appear in nature.

Benner's approach is to create letters that chemically bond to DNA in a similar way to how the natural letters bond. Romesberg, Malyshev and colleagues are working with unnatural letters that are held in place by different forces than those of natural DNA letters. The mechanism is so different than the natural method that the Romesberg team at first feared they had made an error.

According to conventional chemistry, the unnatural letters could not be duplicated by the body's DNA copier, an enzyme called DNA polymerase, because of how they're held in position. However, the Romesberg team found that the enzyme copies the unnatural letters by placing them into the correct position, the so-called "Watson-Crick geometry" that the natural base pairs conform to.